Method for preparation of polyethylene glycol aldehyde derivatives

ABSTRACT

Aldehyde derivatives of polyethylene glycols and methods of making thereof are disclosed. These aldehyde derivatives can be used to make polyethylene glycol-hydrazines, polyethylene glycol-thiols, polyethylene glycol amines, and branched polyethylene glycols. PEG aldehyde derivatives or other functional PEG derivatives prepared from PEG aldehydes are useful for protein conjugation and surface modification.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/105,630, filed Oct. 26, 1998, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

The present invention relates to a direct, very mild, efficient methodof synthesis of derivatives of polyethylene glycol having one or moreterminal aldehyde groups and the use of these derivatives as reagentsfor modifying peptides or proteins and for the preparation of othertypes of functionalized polyethylene glycols.

In the last decade, enormous progress in recombinant DNA technologyenabled discovery and/or production of a large number of physiologicallyactive proteins, many of them having unforeseen potential to be used asbiopharmaceuticals. Unfortunately, most of these peptides and proteinsexhibit very fast plasma clearance, thus requiring frequent injectionsto ensure steady pharmaceutically relevant blood levels of a particularpeptide or protein with pharmacological activity. Further, manypharmaceutically relevant peptides and proteins, even those having humanprimary structure, can be immunogenic, giving rise to production ofneutralizing antibodies circulating in the bloodstream. This isespecially true for intravenous and subcutaneous administration, whichis of particular concern for most peptide and protein drugs.

To solve these problems, various hydrophilic macromolecular compoundshave been conjugated with peptide and protein drugs. This has proven tobe very efficient and useful in decreasing the immunogenicity andincreasing the circulatory half-life of peptide and protein drugs. Amonghydrophilic macromolecular compounds, polyethylene glycol derivativeshave been used most frequently for synthesis of peptide and proteinconjugates, because such derivatives are non-immunogenic and veryhydrophilic, and thus do not affect the three dimensional structure(folding) of protein drugs. Moreover, dynamic polyethylene chains alsoprovide protection against hydrolytic degradation by proteolyticenzymes.

To achieve significant level of modification of peptides and proteinswith polyethylene glycol (PEG) derivatives, several methods ofactivation have been widely used, such as the triazine method (F. F.Davis et al., U.S. Pat. No. 4,179,337), the active ester method withN-hydroxysuccinimide (F. Veronese et al., U.S. Pat. No. 5,286,637), andthe direct activation method with carbonyldiimidazole (G. S. Bethell etal., 254 J. Biol. Chem. 2572-2574 (1979)). Typically, these activatedPEG derivatives contain an electrophilic center that is available forreaction with nucleophilic centers on peptides and proteins, such asamino groups. The common disadvantage of these derivatives isinstability in aqueous media against nonspecific hydrolysis at alkalinepH, where the modification reaction usually takes place. Recently,attachment of PEG chains to peptides and proteins via reductivealkylation has been described (P. Wirth et al., 19 Bioorg. Chem. 133-142(1991); S. M. Chamow et al., 5 Bioconjugate Chem 133-140 (1994); O. B.Kinstler et al., 13 Pharm. Res. 996-1002 (1996)). This method requiresintroduction of an aldehyde group at the end of amonomethoxypolyethylene glycol (MePEG) chain that originally carried ahydroxyl group. Contrary to other activated PEG derivatives mentionedabove, PEG-aldehydes, like aldehydes in general, are mostly inert towater and react selectively with the amino groups of peptides andproteins as nucleophiles in aqueous media. These two properties arehighly desirable, not only because of stability upon long-term storage,but also because of high selectivity for coupling of these aldehyde-PEGderivatives and for simplicity.

Originally, MePEG was oxidized with active MnO₂ to yield MePEG-ethanal(acetaldehyde) as described in U.S. Pat. No. 4,002,531 for attaching PEGchains to enzymes and other proteins. This procedure was later shown tobe very inefficient by M. S. Paley & J. M. Harris, 25 J. Polym. Sci.Polym. Chem. Edn. 2447-2454 (1987). Thus, other oxidative methods, suchas the Moffatt procedure, were introduced as described by Harris et al.,22 J. Polym. Sci. Polym. Chem. Edn. 341-352 (1984). These oxidativereactions, however, are not quantitative and may be accompanied byunwanted side reactions and/or MePEG chain cleavage. Further, productpurification is difficult. In parallel, a more gentle and quantitativealkylation method using β-bromopropionaldehyde has been used to prepareMePEG-aldehyde derivatives (J. M. Harris & M. R.-Sedaghat-Herati, U.S.Pat. No. 5,252,714). A terminal ethyl spacer was introduced to increasethe stability of MePEG aldehyde derivatives in water in the presence ofbase since it was argued, on the basis of CH₃—O—CH₂—CH₂—O—CH₂—CHOchemical stability data (M. S. Paley & J. M. Harris, 25 J. Polym. Sci.Polym. Chem. Edn. 2447-2454 (1987)), that MePEG-ethanal derivatives areunstable in water in the presence of base. To the contrary,MePEG-ethanal derivatives were successfully used for proteinmodification when used in 20 to 200 molar excess (PEG derivative/Proteinmolar ratio) at pH 7.0-8.0 (P. Wirth et al., 19 Bioorg. Chem. 133-142(1991); S. M. Charnow et al., 5 Bioconjugate Chem 133-140 (1994); O. B.Kinstler et al., 13 Pharm. Res. 996-1002 (1996)).

A new catalytic, oxidative method for conversion of alcohols intocarbonyl compounds utilizing oxygen or even air as the ultimate andstoichiometric oxidant, producing water as the only byproduct, has beenrecently discovered and described by I. E. Marko et al., 274 Science2044-2046(1996). The active catalyst appears to be heterogeneous,absorbed on insoluble K₂CO₃, and is composed of CuCl,diethylazodicarboxylate or the corresponding hydrazine, and1,10-phenanthroline. Apolar solvents such as benzene or toluene arerequired. K₂CO₃, besides its role as a solid support, also acts as abase and as a water scavenger, but can be replaced by 4 Å molecularsieves and a catalytic amount of nonoxidizable base, such as KOBu^(t).This process is very efficient under mild conditions (temp. 70-90° C.),providing high degrees of conversion (80-100%), and is not onlyeconomically viable but it is also environmentally friendly.

In view of the foregoing, it will be appreciated that providing anefficient, gentle method for preparation of polyethylene glycol aldehydederivatives would be a significant advancement in the art.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of preparationof PEG-dialdehydes and alkoxyPEG (i.e., RO-PEG) aldehydes and theirhomologues that retain reactivity in water and selectively react withamino groups.

Another object of the invention is to provide PEG-dialdehydes andRO-PEG-aldehydes and their homologuestat react selectively with aminogroups and are stable in water.

Yet another object of the invention is to provide bifunctional PEG andmonofunctional RO-PEG derivatives and their homologues that can beprepared starting from PEG-dialdehydes and RO-PEG-aldehydes or theirhomologues.

Still another object of this invention is the process of preparation ofbifunctional PEG and monofunctional RO-PEG derivatives and theirhomologues starting from PEG-dialdehydes and RO-PEG-aldehydes or theirhomologues.

These and other objects can be addressed by providing compositions,methods of making thereof, and methods of using thereof. An illustrativeembodiment of the invention relates to the oxidation of polyethyleneglycol derivatives having the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 2 to about 12, utilizing a catalytic procedure that provides highyields of corresponding PEG-dialdehyde derivatives and RO-PEG-aldehydederivatives having the formula:

and enables their preparation and isolation in the absence of water.

Another illustrative embodiment of the invention relates to aldehydederivatives of polyethylene glycos of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 4 to about 12.

Still another embodiment of the invention relates to polyethylene glycolderivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 2 to about 12.

Yet another illustrative embodiment of the invention relates topolyethylene glycol derivatives of formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, and p is an integer from about 2 to about 12.

A further illustrative embodiment of the invention relates to methods ofpreparation of the polyethylene glycol derivatives summarized above.Thus, PEG-dihydrazine derivatives and RO-PEG-hydrazine derivatives areprepared by reacting a particular PEG-dialdehyde derivative orRO-PEG-aldehyde derivative with hydrazine in the presence of reducingagent such as NaBH₃CN or NaBH₄. Also, PEG-dithiol derivatives andRO-PEG-thiol derivatives can be synthesized from a particularPEG-dialdehyde derivative or RO-PEG-aldehyde derivative and selectedmercaptoalkylamine in the presence of reducing agent such as NaBH₃CN orNaBH₄.

A still further embodiment of the invention relates to branchedpolyethylene glycol derivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, and r is an integer from 0 to about 5. These branchedpolyethylene glycol derivatives are prepared by reacting a particularRO-PEG-aldehyde derivative with a selected diaminocarboxylic acid, whichdoes not necessarily have to be an α-acarboxylic acid as shown in theformula but also can be β-, γ-, or δ-carboxylic acid, in the presence ofreducing agent such as NaBH₃CN or NaBH₄.

Another illustrative embodiment of the invention relates to branchedpolyethylene glycol derivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, r is an integer from 0 to about 5, and s is 2 or 3. Thistype of branched polyethylene glycol derivative is prepared startingfrom monoaminoPEG derivatives, as set forth below, and reacting themwith dicarboxylic acid cyclic anhydrides, thus converting monoaminoPEGderivatives into monocarboxyPEG derivatives. A selected activatedmonocarboxyPEG derivative is then reacted with a selecteddiaminocarboxylic acid, which does not necessarily have to be ana-carboxylic acid but can also be β-, γ-, or δ-carboxylic, yielding abranched polyethylene glycol derivative as specified by the generalformula above.

Finally, another illustrative embodiment of the invention relates tomethods of preparation of amino-PEG derivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 3 to about 12. These derivatives can be synthesized by reductiveamination of corresponding PEG-dialdehyde derivatives or RO-PEG-aldehydederivatives, prepared as disclosed above, with ammonium salt in thepresence of reducing agent such as NaBH₃CN or NaBH₄.

DETAILED DESCRIPTION

Before the present polyethylene glycol aldehydes, derivatives thereof,and methods for making thereof are disclosed and described, it is to beunderstood that this invention is not limited to the particularconfigurations, process steps, and materials disclosed herein as suchconfigurations, process steps, and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

The publications and other reference materials referred to herein todescribe the background of the invention and to provide additionaldetail regarding its practice are hereby incorporated by reference. Thereferences discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an apolar solvent” includes a mixture of two or moreapolar solvents, reference to “a catalyst” includes reference to one ormore of such catalysts, and reference to “a polyethylene glycol”includes reference to a mixture of two or more polyethylene glycols.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outherein.

As used herein, “comprising,” “including,” “containing,” “characterizedby,” and grammatical equivalents thereof are inclusive or open-endedterms that do not exclude additional, unrecited elements or methodsteps. “Comprising” is to be interpreted as including the morerestrictive terms “consisting of” and “consisting essentially of.”

As used herein, “consisting of” and grammatical equivalents thereofexclude any element, step, or ingredient not specified in the claim.

As used herein, “consisting essentially of” and grammatical equivalentsthereof limit the scope of a claim to the specified materials or stepsand those that do not materially affect the basic and novelcharacteristic or characteristics of the claimed invention.

As used herein, “peptide” means peptides of any length and includesproteins. The terms “polypeptide” and “oligopeptide” are used hereinwithout any particular intended size limitation, unless a particularsize is otherwise stated. Typical of peptides that can be utilized arethose selected from group consisting of oxytocin, vasopressin,adrenocorticotrophic hormone, epidermal growth factor, prolactin,luliberin or luteinising hormone releasing hormone, growth hormone,growth hormone releasing factor, insulin, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastrone, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, gramicidins, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines. The onlylimitation to the peptide or protein that may be utilized is one offunctionality.

In the present specification, the lower alkyl groups represented by R,R₁, and R₂ may be either straight or branched, preferably lower alkylgroups having 1 to 4 carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, and the like. Polyethylene glycolderivatives and monoalkoxypolyethylene glycol derivatives of theformula:

wherein R₁ is lower alkyl, R₂ is lower alkyl or H, n is an integer fromabout 3 to about 500, and k and m are integers from about 2 to about 12,can be easily produced by some of the well-known methods in the art.These PEG derivatives or their homologues are dissolved in an apolarsolvent such as benzene or, preferably, toluene, and 2 equivalents ofK₂CO₃ for each hydroxyl group are added. Also, an effective amount of acatalyst should be added to the reaction mixture. By “effective amount”is meant an amount sufficient to increase the rate of reaction. Such aneffective amount can be determined as a matter of routine by a personskilled in the art. Preferably, the amount of catalyst added to thereaction is about 1 to about 10 mol %, more preferably about 5 mol %,with regard to hydroxyl groups. The catalyst comprises (i) a transitionmetal cation having two main oxidative states, (ii) an aromaticheterocycle containing nitrogen, and (iii) a member selected from thegroup consisting of diethylazodicarboxylate, a hydrazine derivative ofdiethylazodicarboxylate, and tert-butyl analogs thereof. The transitionmetal cation having two main oxidative states is preferably a memberselected from the group consisting of Cu⁺, Co²⁺, Fe²⁺, Ni²⁺, andmixtures thereof. More preferably, the transition metal cation is Cu⁺.Preferred aromatic heterocycles containing nitrogen include1,10-phenanthroline and α,α′-dipyridyl. Preferably, the member selectedfrom the group consisting of diethylazodicarboxylate, a hydrazinederivative of diethylazodicarboxylate, and tert-butyl analogs thereof isdiethylazodicarboxylate. An especially preferred catalyst comprises CuCl(cuprous chloride), 1,10-phenanthroline, and diethylazodicarboxylate.The catalyst is added to the mixture, and O₂ or air (aerobic conditions)is bubbled through the mixture with the temperature of the mixture inthe range of about 40° C. to about 90° C. It is also noteworthy thatcatalyst deactivation is not observed and insoluble catalyst, which caneasily be separated from the rest of reaction mixture, can be recycledand repetitively used in a new reaction. With respect to the compositionof the catalyst, Cu⁺ can be replaced by other transient metal cationshaving two main oxidative states, such as Co²⁺, Fe²⁺, or Ni²⁺.Phenanthroline can be replaced by other aromatic heterocycles containingnitrogen such as α,α′-dipyridyl. Diethylazodicarboxylate or itshydrazine derivative can be replaced by corresponding tert-butylanalogs, which remarkably improve the rate of the reaction and thelifetime of catalyst (I. E. Marko et al., 274 Science 2044-2046 (1996)).

The reaction can be described as follows:

wherein R₁ is lower alkyl, R₂ is lower alkyl or H, n is an integer ofabout 3 to about 500, and k and m are integers of about 2 to about 12.After completion of the reaction and removal of catalyst by filtration,the PEG-aldehyde derivative product can be easily purified and recoveredat room temperature by precipitation in dry ether and filtration of thesolid product, which can be stored in a stable solid form until use. Forlow molecular weight PEG-aldehyde derivatives (600 or lower), the sameprecipitation procedure can be used. In this case, however, theprecipitating ether medium has to be kept at −20° C., and theprecipitated PEG-aldehyde derivative is separated by centrifugation at−20° C.

Type I PEG-aldehyde derivatives are ideal for protein modificationbecause the reaction involved, reductive amination, is highly selectivefor amino groups. Reductive amination is well known in the art, and canbe described by the formula PEG-CHO+NH₂—R′→PEG-CH—NHR′. E.g, R. T.Morrison & R. N. Boyd, Organic Chemistry 735, 740741 (3^(rd) ed., 1973).The reductive amination reaction can be easily performed in waterbecause the RO-PEG-aldehyde derivatives exhibit substantial stability ascompared to other activated PEG derivatives for protein modification,and the extent of the reaction can also be controlled such that partialmodification is possible. The last goal is achieved by varying the molarratio of protein to RO-PEG-aldehyde derivative (usually 5 to 50 molarexcess) and/or pH of the coupling reaction which can Be kept in therange from pH 5.0 to 9.0. Using slightly acidic conditions (pH 5.0-6.0)for coupling, it is possible to selectively target the N-terminal aminogroups of peptides or proteins, since the ε-amino groups on lysineresidues do not react under these conditions because they are fullyprotonated. Concomitantly, RO-PEG-aldehyde derivatives are stable andreactive under slightly acidic conditions. Another advantage of thereductive amination method for PEG chain attachment is that it preservesthe overall charge when primary amino groups are converted intosecondary amino groups.

Type II PEG-aldehyde derivatives can be used for protein immobilization.In this case, a PEG-dialdehyde derivative is first coupled to anamino-group-modified surface in water. Excess PEG-dialdehyde derivativeis then washed away and bound PEG-dialdehyde with one available aldehydegroup is reacted with some other molecule such as a peptide or protein.

Independently, the above described PEG-aldehyde derivatives, either typeI or II, can also be utilized for the preparation of many other usefulPEG derivatives. Thus, by reacting PEG-aldehyde derivatives prepared asdisclosed above with a large excess of hydrazine in dimethyl sulfoxide(DMSO) at room or elevated temperature in the presence of reducingagent, PEG-hydrazine derivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 2 to about 12, can be synthesized. A resulting PEG-hydrazinederivative is purified by precipitation with excess ether and recoveredby filtration or centrifugation. The completion of the coupling reactionis confirmed by FTIR spectroscopy through the absence of CHO specificbands.

In parallel, by reacting PEG-aldehyde derivatives, prepared as describedabove, with a large excess of aminoalkylmercaptans of the formulaNH₂—(CH₂)_(p)—SH, wherein p is an integer from about 2 to about 12, inDMSO, dimethylformamide (DMF), tetrahydrofuran (THF), or dioxane at roomor elevated temperature in the presence of reducing agent, PEG-thiolderivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k, m, and p are integers fromabout 2 to about 12, can be prepared. A resulting PEG-thiol derivativeis purified by precipitation in excess of ether and recovered byfiltration or centrifugation. The completion of the reaction isconfirmed by FTIR spectroscopy through the absence of CHO-specificbands. Further, SH-group content is determined by thiol titration withdipyridyl disulfide at 343 nr by UV spectroscopy.

Also, branched PEG derivatives can be synthesized starting from type IPEG-aldehyde derivatives, as specified above, and diaminocarboxylicacids such as ornithine or lysine at room or elevated temperature in thepresence of reducing agent in the appropriate solvent having theformula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, and r is an integer from 0 to about 5. The relativeconfiguration of two amino groups and a carboxylic group indiaminocarboxylic acid can be different from that shown in the formulaabove. A branched PEG derivative is purified by precipitation in excessether and recovered by filtration. The completion of the reaction isconfirmed by FTIR spectroscopy through the absence of CHO specificbands. Moreover, residual primary amino group content in the preparationis determined by TNBS assay (R. Fields, 25 Methods Enzymol. 464-468(1972)). Such branched PEG derivatives having a single terminalcarboxylic group can be activated through the attachment ofN-hydroxysuccinimide to this carboxyl group, thus forming an activeester. Such N-hydroxysuccinimide ester of a particular branched PEGderivative can then be reacted with a selected peptide or protein at pH7.0-9.0 in aqueous buffer to prepare a particular branched PEG-peptideor PEG-protein conjugate as described in the open literature (R. Clarket al., 271 J. Biol. Chem. 21969-210977 (1996); C. Manfardini et al., 6Bioconjugate Chem. 62-69 (1995)). This specific modification procedurewill make the protein conjugate more basic as compared to native protein(one lysine &-amino group is discharged by amide bond formation but twosecondary amino groups are introduced instead), and will thus increaseits solubility at pH 7.4 even without taking into account thesolubilizing effect of the attached PEG chains.

Also, amino-PEG derivatives can be synthesized starting from type I ortype II PEG-aldehyde derivatives, as specified above, and 10 molarexcess of ammonium acetate or ammonium chloride at room or elevatedtemperature in the presence of base such as KOH in methanol. Afterstirring for 1 hour or less, reducing agent such as NaBH₃CH or NaBH₄(also dissolved in methanol) is added, as described for classicalpolyethylene glycol aldehydes by J. M. Harris et al., 22 J. Polym. Sci.Polym. Chem. Edn. 341-352 (1984). The resulting amino-PEG derivativeshave the formula:

wherein R₁ represents lower alkyl R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 3 to about 12. A resulting amino-PEG derivative is purified byprecipitation in excess ether and recovered by filtration orcentrifugation. The completion of the reaction is monitored by FTIRthrough the absence of CHO specific bands. Further, primary amino groupcontent in the resulting amino-PEG derivative is determined by TNBSassay. These amino-PEG derivatives, especially those having one R₁O endgroup, can be reacted with succinic or glutaric anhydride yieldingsuccinamido-carboxyl or glutaramido- carboxyl PEG derivatives. Theintroduced carboxylic group can be activated by some of the well-knownmethods in the art, such as conversion to an acid chloride, R. T.Morrison & R. N. Boyd, Organic Chemistry 590, 601 (₃ ^(rd) ed., 1973),and such activated carboxyl-PEG derivatives can be reacted with aparticular peptide or protein to prepare a particular PEGderivative-peptide or PEG-derivative-protein conjugate of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, s is 2 or 3, and P is a protein or peptide. The number ofPEG derivative chains coupled to a protein or peptide molecule can varydepending on the number of lysine ε-amino groups available andprotein/PEG-derivative molar ratio in the modification reaction.Alternatively, such activated carboxyl-PEG derivatives can be reactedwith a diaminocarboxylic acids as specified above to prepare branchedPEG derivatives of the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, k and m are integers from about 2to about 12, r is an integer from 0 to about 5, and s is 2 or 3. Therelative configuration of two amino groups and a carboxylic group indiaminocarboxylic acid can be different from that shown in the formulaabove. These branched PEG derivatives can also be used after singlecarboxylic group activation by some of the well-known methods in the artfor the synthesis of branched PEG derivative-peptide or PEGderivative-protein conjugates. Again, the number of branched PEGderivatives attached can vary depending on the number of lysine ε-aminogroups available and protein/branched PEG derivative molar ratio in thecoupling reaction.

What is claimed is:
 1. A method for preparing a PEG aldehyde having theformula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 2 to about 12, comprising the steps of: (a) dissolving a PEGderivative having the formula:

wherein R₁ represents lower alkyl, R₂ represents lower alkyl or H, n isan integer from about 3 to about 500, and k and m are integers fromabout 2 to about 12, in an apolar solvent containing 2 equivalents ofK₂CO₃ for each hydroxyl group to form a mixture; (b) adding an effectiveamount of a catalyst and O₂ to the mixture, heating at 40-90° C., andincubating for a sufficient period of time for the PEG derivative to beoxidized to the PEG aldehyde; (c) removing the catalyst; and (d)recovering the PEG aldehyde.
 2. The method of claim 1 wherein saidapolar solvent is toluene.
 3. The method of claim 1 wherein said apolarsolvent is benzene.
 4. The method of claim 1 wherein said catalystcomprises (i) a transition metal cation having two main oxidativestates, (ii) an aromatic heterocycle containing nitrogen, and (iii) amember selected from the group consisting of diethylazodicarboxylate, ahydrazine derivative of diethylazodicarboxylate, and tert-butyl analogsthereof.
 5. The method of claim 4 wherein said transition metal cationhaving two main oxidative states is a member selected from the groupconsisting of Cu⁺, Co²⁺, Fe²⁺, Ni²⁺, and mixtures thereof.
 6. The methodof claim 5 wherein said transition metal cation having two mainoxidative states is Cu⁺.
 7. The method of claim 4 wherein said aromaticheterocycle containing nitrogen comprises 1,10-phenanthroline.
 8. Themethod of claim 4 wherein said aromatic heterocycle containing nitrogencomprises α,α′-dipyridyl.
 9. The method of claim 4 wherein said memberselected from the group consisting of diethylazodicarboxylate, ahydrazine derivative of diethylazodicarboxylate, and tert-butyl analogsthereof is diethylazodicarboxylate.
 10. The method of claim 4 whereinsaid catalyst comprises CuCl, 1,10-phenanthroline, anddiethylazodicarboxylate.
 11. The method of claim 1 wherein saideffective amount of catalyst comprises about 1 to about 10 mol %. 12.The method of claim 11 wherein said effective amount of catalystcomprises about 5 mol %.